The ability to induce bone formation and growth is an area of scientific inquiry which has seen a great deal of activity over the past 20-25 years. Initial observations by Urist et al, Science 150: 893-899 (1965), J. Dent. Res. 50: 1392-1406 (1971), that a substance residing in decalcified bone and now referred to as "bone morphogenic protein" ("BMP" hereafter) could induce ectopic cartilage and bone formation via endochonal ossification after implantation adjacent to skeletal muscles and away from preexisting bone have led to research to isolate and to characterize this material, as well as to identify sources for it. Efforts have also been directed to identifying and isolating other materials having the same or similar properties. For example, a material herein referred to as "BIA" for bone inducing agent has been shown to exist in some strains of transformed, cultured human epithelial and osteosarcoma cells. Exemplary of this are Anderson et al. Am. J. Path. 44:507-519 (1964) and J. Cell Biol. 33: 165-177 (FL transformed amnion cells); Anderson, et al, Fed; Proc; 27: 475 (1868) (HeLa cells); Wlodarski Exp; Cell Res; 57: 446-448 (1969) (WISH amnion cells); Wlodarski, et al Calcif. Tiss. Res. 7: 345-352 (1971); (neoplastic cells) Amitani et al., Gann 66: 327-329 (1975); Hanamura, et al., Clin. Orthop. & Rel. Res. 148: 274-280 (1980) (Dunn mouse osteosarcoma cells of BFO strain).
The patent literature in this field as it relates to BMP is growing. U.S. Pat. Nos. 4,294,753; 4,455,256; 4,526,909; 4,563,489; 4,596,574; 4,619,989; and 4,761,471, all issued to Urist, arise out of the work he has done on BMP. The most recent of these patents describes the isolation of BMP from frozen bone samples isolated from cadavers. Essentially, the bone was frozen, defatted, demineralized and freeze dried, after which a series of dissolving and dialyzing steps were carried out, ending in fractionation, dialysis, and SDS-PAGE analysis. The BMP is described as an acidic protein, with molecular weight of 17.5.+-.0.5 kd for the human variety, whereas bovine BMP has molecular weight of 18.5.+-.0.5 kd. The patent discusses how it is difficult to separate the bovine BMP from associated proteins, and how the associated proteins for both human and bovine BMP are necessary to optimize the effect of the protein on bone growth.
U.S. Patents have issued to others, as exemplified by U.S. Pat. Nos. 4,394,370; 4,472,840; 4,563,350; 4,642,120; and 4,968,590. For the most part, these patents deal with grafts for repairing osseous injuries. The '590 patent describes "pure mammalian osteogenic protein" and its uses. The protein, when glycosylated, has a molecular weight of 30 kd on SDS-PAGE, and is obtained from demineralized bone matrix. The bones are obtained from cattle. This protein may be identical to osteogenin, as described by Luyten et al, J. Biochem. 264: 13377-13380 (1989).
As the field has grown, it is convenient to refer to the class of proteins which have the specified effect as "BMPs". Thus, Wozney et al, Science 242: 1528-1534 (1989), have isolated genes for 3 BMPs, having molecular weights of 30, 18, and 16 kds. Recombinant BMPs have been produced via transfecting these genes into cultured monkey and hamster cells. Wang et al, Proc. Natl. Acad. Sci USA 87: 2220-2224 (1990), describe bone induction in animal when BMP-2A is implanted in GuHCl inactivated, decalcified bone matrix carriers. Sampath et al, J. Biol. Chem. 265: 13198-13205 (1990), describe "osteoinductive protein" (OP), also isolated from demineralized bovine bone. The OP is a 30 kd dimer, having 16+18 kd subunits. This protein also induces bone when implanted with a GuHCl inactivated bone matrix carrier. Bentz et al, J. Biol. Chem 264: 20805-20810 (1989) have described "osteoinductive factor" (OIF) from bovine bone, and characterize it as a 22-28 kd protein which must react with TGF-beta to have osteoinductive effect in vivo. Bessho et al, Biochem. Biophys. Res. Comm. 165: 595-601 (1989), also isolated an osteoinductive protein of 18 kd.
Isolating protein from bone is an onerous task, requiring enormous amounts of raw materials for extremely small yields. The amount of processing involved cuts the available yield as well. Further, the source of the raw material (bone), must generally be animal rather than human. Given the diversity of individuals within an animal species, non-uniformity of protein from batch to batch is to be expected albeit being undesirable.
Given the drawbacks discussed supra, the use of cultured cells as a source of bone inducing agents and/or bone morphogenic proteins presents an attractive alternative. Cell masses can be grown up quickly, and when a cell line is used, one expects the protein to be uniform, since cell lines by definition are uniform.
The aforementioned epithelial cell lines, as indicated, are osteoinductive, but they are osteoinductive only when live cells are injected. The experiments showing this demonstrated efficacy using immunosuppressed animals. See Anderson, Connect. Tiss. Res. 24: 3-12 (1990); Anderson, et al., Am. J. Patho 44: 507-519 (1964); Wlodarski, et al., Calcif. Tiss. Res. 5: 70-79 (1970); Anderson et al, Fed. Proc. 27: 475 (1968). Implantation of live, foreign cells into a subject with a healthy or non-suppressed immune system will almost inevitably result in a strong, perhaps long term immunological response, so administration of live cells is not advisable. Also, when the cell line is tumorigenic, as most cell lines are, the ramifications of administering live cells include serious risks of transplanting neoplasia into the subject. Monitoring the site of implantation with removal at a "critical point" is not possible, due to the risk of malignant invasion by the implant. The immediate suggestion, i.e., to introduce dead epithelial cells killed by freezing repeatedly into the subject, has proven to be ineffective. Devitalized, freeze dried, FL, WISH, and HeLa cells, as well as their extracts, have not been effective as osteoinducing agents. Anderson et al, Clin. Orthop. Rel. Res. 119: 211-224 (1976).
It is rarely the case that one can obtain an osteoinductive cell line which has an effect when the cell line is devitalized. Workers out of the lab of Amitani and Takaoka, as per Amitani et al, Gann 66: 327-329 (1975); Takaoka, et al., Clin Orthop. Rel. Res. 164: 265-270 (1982); Takaoka, et al., Clin Orthop. Rel. Res. 144: 258-264 (1989), have reported positive results with an osteoinductive BFO strain of Dunn murine osteosarcoma, and human cell line H-OS-6. The aforementioned laboratory has not made these cell lines available to other researchers, so this work cannot be verified or compared to results with other cell lines. Also, as the art well knows, the odds against securing a second cell line with properties identical to a first one are astronomical. To be denied access to a cell line, even if one is aware of its lineage, renders it impossible for the artisan to repeat reported work. Even in the case where a researcher reports a detailed, careful protocol for how a cell line was produced, isolated or derived, repetition of this work hardly guarantees reproduction of the initial results. Hence, the existence of cell banks, depositories, and the general cordiality of researchers in sharing cell lines.
When treating humans, it is desirable to treat with material as close to native human material as possible. U.S. Pat. No. 5,035,901, to Anderson at al., describes that crude extracts of cell line Saos-2 induce bone formation, even in devitalized form. The results reported therein were surprising because (i) the prior publications in the field suggested that dead human cell lines were not osteoinductive, and (ii) comparison with many other cell lines showed that the properties of Saos-2 were not shared, either among other human cell lines or among other mammalian cell lines.
The work on Saos-2 has continued. In U.S. patent application Ser. No. 683,283, filed Apr. 10, 1991 extracts of Saos-2 not only possess osteoinductive effect but show activity superior to that possessed by the whole cells. This is surprising because the BIA from Saos-2 cell line has not yet been isolated or characterized. Extraction protocols are known to damage some molecules, and in the process of extraction, one inevitably loses a portion of the starting material. With this lack of certainty in mind, it was thus more than slightly surprising that an Saos- 2 extract, prepared as described infra, was effective in inducing bone formation in subject animals (i.e., rats) that were immunocompetent and have rejected less purified preparations of BIA.
Further purification has now been carried out, and it has been found that extracts of Saos-2 contain, at the least, "bone morphogenic protein 4", or "BMP4" as it is referred to hereafter, as well as transforming growth factor beta (TGF-.beta.). Surprisingly, these materials are present in the non-bone-inducing human osteosarcoma cell line designated U2-OS, but in U2-OS cells are insufficient to provoke the bone inducing effect achieved with Saos-2, indicating that the Saos-2 cell line harbors at least a third, unidentified factor which, in combination with the listed materials, leads to the surprising properties characteristic of Saos-2 extracts. These new extracts are the subject of the invention and are described in the following disclosure.